We have observed that the insulin receptor (IR) content, as measured by 3 different techniques; specific radioimmunoassay (IR-RIA), ligand binding assay, and immunostaining is 6-fold elevated in breast cancers when compared to normal breast tissue. These findings may have important clinical relevance therefore since insulin increases the proliferation of breast cancer cells both in vitro and in vivo. In this proposal, we plan to carry out basic and clinical studies to assess the biological significance of these observations. We plan three basic studies: First, we will investigate the mechanisms of IR overexpression both in breast tumor specimens and breast cancer cell lines by correlating IR protein content, IR mRNA levels, and DNA copy number. We have in vitro evidence that one group of hormones which regulate breast epithelial cells, the progestins, also increase IR gene expression. We will now explore the possibility that certain polypeptide growth factors, which are known to be produced in either human breast tumors or adjacent tissue (TGFs, IGFs), modulate IR expression. Second, the role of IR overexpression in ligand induced malignant transformation will be examined by IR transfection and overexpression in non-transformed breast epithelial cells. In concert with these studies, an IR cDNA expression vector with mammary gland specific promoters will be used to produce transgenic mice to investigate the development of breast tumors. Third, we will isolate a new atypical insulin binding receptor in breast cancer cells and tissues that binds both insulin and IGF-I with high affinity. In clinical studies, we will analyze the prognostic value of IR overexpression as a breast tumor marker. For retrospective studies, we will use the formalin-fixed archive of over 300 tumor specimens in collaboration with Dr. Ann Thor (Harvard). This archive contains specimens from tumors with known clinical outcome (8 year followup) and have been used successfully in correlation studies of other tumor proteins. We will also carry out a prospective study of the specimens we have been collecting and analyzing from the University of Catania, Italy, and the University of California, San Francisco. These clinical and basic studies therefore should provide important information concerning the role of the IR in the biology of breast cancers.